Sphingosine-1-phosphate Secretion Research Articles

β1-Blockade Prevents Post-Ischemic Myocardial Decompensation Via β3AR-Dependent Protective Sphingosine-1 Phosphate Signaling

BackgroundAlthough β-blockers increase survival in patients with heart failure (HF), the mechanisms behind this protection are not fully understood, and not all patients with HF respond favorably to them. We recently showed that, in cardiomyocytes, a reciprocal down-regulation occurs between β1-adrenergic receptors (ARs) and the cardioprotective sphingosine-1-phosphate (S1P) receptor-1 (S1PR1). ObjectivesThe authors hypothesized that, in addition to salutary actions due to direct β1AR-blockade, agents such as metoprolol (Meto) may improve post–myocardial infarction (MI) structural and functional outcomes via restored S1PR1 signaling, and sought to determine mechanisms accounting for this effect. MethodsWe tested the in vitro effects of Meto in HEK293 cells and in ventricular cardiomyocytes isolated from neonatal rats. In vivo, we assessed the effects of Meto in MI wild-type and β3AR knockout mice. ResultsHere we report that, in vitro, Meto prevents catecholamine-induced down-regulation of S1PR1, a major cardiac protective signaling pathway. In vivo, we show that Meto arrests post-MI HF progression in mice as much as chronic S1P treatment. Importantly, human HF subjects receiving β1AR-blockers display elevated circulating S1P levels, confirming that Meto promotes S1P secretion/signaling. Mechanistically, we found that Meto-induced S1P secretion is β3AR-dependent because Meto infusion in β3AR knockout mice does not elevate circulating S1P levels, nor does it ameliorate post-MI dysfunction, as in wild-type mice. ConclusionsOur study uncovers a previously unrecognized mechanism by which β1-blockers prevent HF progression in patients with ischemia, suggesting that β3AR dysfunction may account for limited/null efficacy in β1AR-blocker–insensitive HF subjects.

Role of Sphingosine-1-Phosphate in Mast Cell Functions and Asthma and Its Regulation by Non-Coding RNA.

Sphingolipid metabolites are emerging as important signaling molecules in allergic diseases specifically asthma. One of the sphingolipid metabolite, sphingosine-1-phosphate (S1P), is involved in cell differentiation, proliferation, survival, migration, and angiogenesis. In the allergic diseases, alteration of S1P levels influences the differentiation and responsiveness of mast cells (MCs). S1P is synthesized by two sphingosine kinases (SphKs), sphingosine kinase 1, and sphingosine kinase 2. Engagement of IgE to the FcεRI receptor induces the activation of both the SphKs and generates S1P. Furthermore, SphKs are also essential to FcεRI-mediated MC activation. Activated MCs export S1P into the extracellular space and causes inflammatory response and tissue remodeling. S1P signaling has dual role in allergic responses. Activation of SphKs and secretion of S1P are required for MC activation; however, S1P signaling plays a vital role in the recovery from anaphylaxis. Several non-coding RNAs have been shown to play a crucial role in controlling the MC-associated inflammatory and allergic responses. Thus, S1P signaling pathway and its regulation by non-coding RNA could be explored as an exciting potential therapeutic target for asthma and other MC-associated diseases.

Synergistic Action of Genistein and Calcitriol in Immature Osteosarcoma MG-63 Cells by SGPL1 Up-Regulation.

BackgroundPhytoestrogens such as genistein, the most prominent isoflavone from soy, show concentration-dependent anti-estrogenic or estrogenic effects. High genistein concentrations (>10 μM) also promote proliferation of bone cancer cells in vitro. On the other hand, the most active component of the vitamin D family, calcitriol, has been shown to be tumor protective in vitro and in vivo. The purpose of this study was to examine a putative synergism of genistein and calcitriol in two osteosarcoma cell lines MG-63 (early osteoblast), Saos-2 (mature osteoblast) and primary osteoblasts.MethodsThus, an initial screening based on cell cycle phase alterations, estrogen (ER) and vitamin D receptor (VDR) expression, live cell metabolic monitoring, and metabolomics were performed.ResultsExposure to the combination of 100 μM genistein and 10 nM calcitriol reduced the number of proliferative cells to control levels, increased ERß and VDR expression, and reduced extracellular acidification (40%) as well as respiratory activity (70%), primarily in MG-63 cells. In order to identify the underlying cellular mechanisms in the MG-63 cell line, metabolic profiling via GC/MS technology was conducted. Combined treatment significantly influenced lipids and amino acids preferably, whereas metabolites of the energy metabolism were not altered. The comparative analysis of the log2-ratios revealed that after combined treatment only the metabolite ethanolamine was highly up-regulated. This is the result: a strong overexpression (350%) of the enzyme sphingosine-1-phosphate lyase (SGPL1), which irreversibly degrades sphingosine-1-phosphate (S1P), thereby, generating ethanolamine. S1P production and secretion is associated with an increased capability of migration and invasion of cancer cells.ConclusionFrom these results can be concluded that the tumor promoting effect of high concentrations of genistein in immature osteosarcoma cells is reduced by the co-administration of calcitriol, primarily by the breakdown of S1P. It should be tested whether this anti-metastatic pathway can be stimulated by combined treatment also in metastatic xenograft mice models.

Paradoxical Association of Postoperative Plasma Sphingosine-1-Phosphate with Breast Cancer Aggressiveness and Chemotherapy.

Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that has been shown to serve an important regulatory function in breast cancer progression. This study analyzes plasma S1P levels in breast cancer patients undergoing adjuvant therapy as compared to healthy control volunteers. 452 plasma S1P samples among 158 breast cancer patients, along with 20 healthy control volunteers, were analyzed. Mean S1P levels did not significantly differ between cancer patients and controls. Smoking was associated with higher S1P levels in cancer patients. Baseline S1P levels had weak inverse correlation with levels of the inflammatory mediator interleukin- (IL-) 17 and CCL-2 and positive correlation with tumor necrosis factor alpha (TNF-α). Midpoint S1P levels during adjuvant therapy were lower than baseline, with near return to baseline after completion, indicating a relationship between chemotherapy and circulating S1P. While stage of disease did not correlate with plasma S1P levels, they were lower among patients with Her2-enriched and triple-negative breast cancer as compared to luminal-type breast cancer. Plasma S1P levels are paradoxically suppressed in aggressive breast cancer and during adjuvant chemotherapy, which raises the possibility that postoperative plasma S1P levels do not reflect S1P secretion from resected breast cancer.

Abstract P2-05-11: Sphingosine-1-phosphate signaling promotes metastatic niches and lung metastasis in obesity-related breast cancer

Abstract INTRODUCTION: The link between obesity and elevated breast cancer mortality is well known, however, the underlying mechanisms are poorly understood. Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator produced by sphingosine kinases (SphKs) that plays critical roles in inflammation and cancer progression. Previously, we found that obesity increases levels of S1P not only in breast tumors, but also in the lung. "Metastatic niches" are specialized microenvironments in distant organs primed by factors from cancer cells. We hypothesized that S1P secreted from the primary tumor could promote formation of a "metastatic niche" in the lung, which assists circulating cancer cells to form metastatic lesions. Further, HFD-induced obesity increases S1P secretion from the primary tumor, which could promote the formation of "metastatic niches" in the lung and lung metastasis. The aim of this study is to test these hypotheses. METHODS: A mouse model utilizing tail vein injection of E0771 syngeneic breast cancer cells was used. Prior to tail vein injections of naive E0771 cells, mice were treated with conditioned media from E0771 breast cancer cells overexpressing SphK1 (K1-CM) or that from E0771 cells cultured with the vector control (CT-CM). Histological analysis, RT-qPCR, and western blot were used. RESULTS: The lungs after K1-CM treatment demonstrated much more infiltration of macrophages with greater IL-6 secretion than lungs from CT-CM mice in areas without metastasis. Furthermore, SphK1, S1P receptor 1 and IL-6 expression were all significantly higher in the lungs of mice treated with K1-CM than with CT-CM, suggesting that S1P secreted from the primary tumor promotes formation of a metastatic niche in the lung. Next, mice were fed with HFD or ND for 12 weeks before treatment with SphK1-CM, and lungs were examined 7 days after intravenous injection of E0771 cells. Histological analysis demonstrated that there were significantly more lung metastases in mice on HFD than in mice on ND. Importantly, treatment with FTY720, a functional antagonist of S1P receptor 1, significantly reduced the lung metastases in HFD fed animals. Immunofluorescent staining revealed higher expression of IL-6 and greater number of F4/80 positive macrophages in mice fed with HFD compared with mice fed with ND, whereas FTY720 dramatically suppressed both IL-6 and macrophage infiltration in the lung of HFD-fed mice. HFD-induced obesity also increased pERK, pAKT, pStat3, and pp65 in the lung, and FTY720 suppressed these signaling pathways. CONCLUSION: Our results suggest that S1P plays a role in the formation of "metastatic niches" in the lung and lung metastasis of breast cancer, and obesity promotes this process. S1P will be a promising target for treatment of breast cancer metastasis, especially in condition with obesity. This work was supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research Grant Number 15H05676 and 15K15471 for M.N and 15H04927 for W.T. M.N. is supported by the Uehara Memorial Foundation, Nakayama Cancer Research Institute, and Tsukada Medical Foundation. K.T. is supported by NIH/NCI grant R01CA160688 and Susan G. Komen Investigator Initiated Research Grant IIR12222224. Citation Format: Nagahashi M, Tsuchida J, Moro K, Tatsuda K, Koyama Y, Takabe K, Wakai T. Sphingosine-1-phosphate signaling promotes metastatic niches and lung metastasis in obesity-related breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-11.

Uncleaved ApoM Signal Peptide Is Required for Formation of Large ApoM/Sphingosine 1-Phosphate (S1P)-enriched HDL Particles

Apolipoprotein M (apoM), a plasma sphingosine 1-phosphate (S1P) carrier, associates with plasma HDL via its uncleaved signal peptide. Hepatocyte-specific apoM overexpression in mice stimulates formation of both larger nascent HDL in hepatocytes and larger mature apoM/S1P-enriched HDL particles in plasma by enhancing hepatic S1P synthesis and secretion. Mutagenesis of apoM glutamine 22 to alanine (apoM(Q22A)) introduces a functional signal peptidase cleavage site. Expression of apoM(Q22A) in ABCA1-expressing HEK293 cells resulted in the formation of smaller nascent HDL particles compared with wild type apoM (apoM(WT)). When apoM(Q22A) was expressed in vivo, using recombinant adenoviruses, smaller plasma HDL particles and decreased plasma S1P and apoM were observed relative to expression of apoM(WT). Hepatocytes isolated from both apoM(WT)- and apoM(Q22A)-expressing mice displayed an equivalent increase in cellular levels of S1P, relative to LacZ controls; however, relative to apoM(WT), apoM(Q22A) hepatocytes displayed more rapid apoM and S1P secretion but minimal apoM(Q22A) bound to nascent lipoproteins. Pharmacologic inhibition of ceramide synthesis increased cellular sphingosine and S1P but not medium S1P in both apoM(WT) and apoM(Q22A) hepatocytes. We conclude that apoM secretion is rate-limiting for hepatocyte S1P secretion and that its uncleaved signal peptide delays apoM trafficking out of the cell, promoting formation of larger nascent apoM- and S1P-enriched HDL particles that are probably precursors of larger apoM/S1P-enriched plasma HDL.

Export of sphingosine-1-phosphate and cancer progression

Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that promotes cell survival, proliferation, migration, angiogenesis, lymphangiogenesis, and immune response; all are critical processes of cancer progression. Although some important roles of intracellular S1P have recently been uncovered, the majority of its biological effects are known to be mediated via activation of five specific G protein-coupled receptors [S1P receptor (S1PR)1-S1PR5] located on the cell surface. Secretion of S1P produced inside cells by sphingosine kinases can then signal through these receptors in autocrine, paracrine, and/or endocrine manners, coined "inside-out" signaling of S1P. Numerous studies suggest that secreted S1P plays important roles in cancer progression; thus, understanding the mechanism by which S1P is exported out of cells, particularly cancer cells, is both interesting and important. Here we will review the current understanding of the transport of S1P out of cancer cells and its potential roles in the tumor microenvironment.

Osteoblast-derived sphingosine 1-phosphate to induce proliferation and confer resistance to therapeutics to bone metastasis-derived prostate cancer cells

Sphingosine 1-phosphate (S1P) plays important roles in cell proliferation, differentiation or survival mainly through its surface G-protein-coupled receptors S1P1−5. Bone represents the major site of metastasis for prostate cancer (CaP) cells, which rely on bone-derived factors to support their proliferation and resistance to therapeutics.In the present work we have found that conditioned medium (CM) from the MC3T3 osteoblastic cell line or primary murine and human osteoblast-like cells, as well as co-culture with MC3T3 stimulate proliferation of CaP lines in S1P-dependent manner. In addition, osteoblastic-derived S1P induces resistance of CaP cells to therapeutics including chemotherapy and radiotherapy. When S1P release from osteoblastic cells is decreased (inhibition of SphK1, knock-down of SphK1 or the S1P transporter, Spns2 by siRNA) or secreted S1P neutralized with anti-S1P antibody, the proliferative and survival effects of osteoblasts on CaP cells are abolished. Because of the paracrine nature of the signaling, we studied the role of the S1P receptors expressed on CaP cells in the communication with S1P secreted by osteoblasts. Strategies aimed at down-regulating S1P1, S1P2 or S1P3 (siRNA, antagonists), established the exclusive role of the S1P/S1P1 signaling between osteoblasts and CaP cells.Bone metastases from CaP are associated with osteoblastic differentiation resulting in abnormal bone formation. We show that the autocrine S1P/S1P3 signaling is central during differentiation to mature osteoblasts by regulating Runx2 level, a key transcription factor involved in osteoblastic maturation. Importantly, differentiated osteoblasts exhibited enhanced secretion of S1P and further stimulated CaP cell proliferation in a S1P-dependent manner.By establishing the dual role of osteoblast-borne S1P on both osteoblastic differentiation and CaP cell proliferation and survival, we uncover the importance of S1P in the bone metastatic microenvironment, which may open a novel area of study for the treatment of CaP bone metastasis by targeting S1P.

Hepatic Apolipoprotein M (ApoM) Overexpression Stimulates Formation of Larger ApoM/Sphingosine 1-Phosphate-enriched Plasma High Density Lipoprotein

Apolipoprotein M (apoM), a lipocalin family member, preferentially associates with plasma HDL and binds plasma sphingosine 1-phosphate (S1P), a signaling molecule active in immune homeostasis and endothelial barrier function. ApoM overexpression in ABCA1-expressing HEK293 cells stimulated larger nascent HDL formation, compared with cells that did not express apoM; however, the in vivo role of apoM in HDL metabolism remains poorly understood. To test whether hepatic apoM overexpression increases plasma HDL size, we generated hepatocyte-specific apoM transgenic (APOM Tg) mice, which had an ∼3-5-fold increase in plasma apoM levels compared with wild-type mice. Although HDL cholesterol concentrations were similar to wild-type mice, APOM Tg mice had larger plasma HDLs enriched in apoM, cholesteryl ester, lecithin:cholesterol acyltransferase, and S1P. Despite the presence of larger plasma HDLs in APOM Tg mice, in vivo macrophage reverse cholesterol transport capacity was similar to that in wild-type mice. APOM Tg mice had an ∼5-fold increase in plasma S1P, which was predominantly associated with larger plasma HDLs. Primary hepatocytes from APOM Tg mice generated larger nascent HDLs and displayed increased sphingolipid synthesis and S1P secretion. Inhibition of ceramide synthases in hepatocytes increased cellular S1P levels but not S1P secretion, suggesting that apoM is rate-limiting in the export of hepatocyte S1P. Our data indicate that hepatocyte-specific apoM overexpression generates larger nascent HDLs and larger plasma HDLs, which preferentially bind apoM and S1P, and stimulates S1P biosynthesis for secretion. The unique apoM/S1P-enriched plasma HDL may serve to deliver S1P to extrahepatic tissues for atheroprotection and may have other as yet unidentified functions.

Abstract P6-06-27: Expression of ATP-binding cassette transporter C1 (ABCC1) and activated sphingosine kinase 1 in breast cancer are associated with significantly shorter disease free survival

Abstract Background and specific objectives: ATP-binding cassette (ABC) transporters are known to be multidrug resistance proteins that efflux various compounds out of cells including chemotherapeutic agents. A number of clinical trails have been conducted targeting ABCB1: however, none showed beneficial effects. The pleiotropic bioactive lipid mediator sphingosine-1-phosphate (S1P), which is generated by sphingosine kinase 1 (SphK1) inside breast cancer cells and exerts its functions by binding to its specific cell surface G-protein coupled receptors (S1PR1-5) after being exported, is now known as a key regulatory molecule in breast cancer progression. We have previously demonstrated that ABCC1 and ABCG2, but not ABCB1 export S1P out of MCF7 human breast cancer cells. We hypothesized that ABCC1 expression in the presence of S1P produced by activated SphK1 in human breast cancer is associated with poor prognosis. Methods: We constructed a tissue microarray with 281 breast tumors from patients, and analyzed expressions of ABCB1, ABCC1, and ABCG2, activated SphK1 (pSphK1), and S1P receptor-1 (S1PR1) immunohistochemically. Breast cancer subtypes were determined by immunohistochemistry of ER, PR, and HER2. Protein expressions were correlated to clinicopathological characteristics, clinical follow-up, and pathological complete response to neoadjuvant chemotherapy. For in vitro experiments, MCF7 human breast cancer cells were transfected with ABCB1 or ABCC1 and stimulated with estradiol. Cell proliferation was analyzed by WST-8 assay. Results: The tissue microarray was comprised of 191 luminal A (68.0%), 17 luminal B (6.0%), 27 HER2 (9.6%), and 46 triple-negative (16.4%) tumors. Activated SphK1 was highly expressed in the patients with lymph node metastasis (40.1% vs 27.3%, P = 0.037) and the pSphK1 high expression group had significantly shorter disease free survival (DFS) (P = 0.05). Eighty percent of the patients expressed S1PR1; however, there were no significant differences in prognosis. On the other hand, ABCC1 expression was associated with significantly shorter DFS (P = 0.027). ABCC1 and ABCG2, but not ABCB1, were significantly higher and more frequently expressed in aggressive subtypes. Patients with tumors expressing both pSphK1 and ABCC1 had significantly shorter DFS (P = 0.002), while patients expressing both ABCB1 and pSphK1 did not. Overexpression of ABCC1 in MCF7 cells not only increased S1P secretion, it significantly increased estradiol-dependent proliferation, compared to MCF7 cells transfected with control vector or ABCB1 (P = 0.010 and P = 0.027, respectively). Conclusions: We have shown that ABCC1 and ABCG2 are highly expressed in aggressive breast cancer subtypes, and that co-expression of pSphK1 and ABCC1 in the tumors is associated with poor prognosis. Our results suggest that inside-out signaling of S1P via ABCC1 may play a significant role in the course of human breast cancer progression. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-06-27.

Abstract 19183: Overexpression of Apom in Liver Stimulates Formation of Larger, Apom/S1P-Enriched Plasma HDL

Apolipoprotein M (ApoM), a lipocalin family member, preferentially associates with plasma HDL and binds plasma sphingosine 1-phosphate (S1P), a critical signaling molecule in immune homeostasis and endothelial barrier function. ApoM overexpression in ABCA1-expressing HEK293 cells stimulates larger nascent HDL particle formation compared to non apoM-expressing cells, but the in vivo role of apoM in HDL metabolism is poorly understood. To test whether hepatic apoM overexpression increases plasma HDL size, we generated liver-specific apoM transgenic (apoM Tg) mice displaying ~5-fold increase in plasma apoM levels compare to wild type (WT) mice. Although plasma HDL cholesterol concentrations were similar to WT mice, apoM Tg mice had larger plasma HDL particles, enriched in apoM, cholesteryl ester, lecithin:cholesterol acyltransferase and S1P. Despite the presence of larger plasma HDL particles in apoM Tg mice, in vivo macrophage reverse cholesterol transport capacity was similar to WT mice. ApoM Tg mice had ~5-fold increase in plasma S1P that was predominantly associated with larger plasma HDL particles. Primary hepatocytes from apoM Tg vs. WT mice generated larger nascent HDL particles, stimulated sphingolipid synthesis, and increased secretion of S1P. Chemical inhibition of ceramide synthase in hepatocytes stimulated S1P synthesis, but not secretion, suggesting that apoM is rate limiting in export of hepatocyte S1P. Our data suggest that liver-specific apoM overexpression: 1) generates large nascent HDLs and large plasma HDL particles that preferentially bind apoM and S1P, and 2) stimulates S1P synthesis for secretion into plasma. The unique apoM/S1P-enriched plasma HDL may serve to deliver S1P to extrahepatic tissues for atheroprotection or other as yet unidentified functions.

Sphingosine-1-Phosphate: a Master Regulator of Lymphocyte Egress and Immunity

Sphingosine-1-phosphate (S1P) is a central factor responsible for lymphocyte distribution in the body. S1P is able to control the integrity of various effector cell populations within many lymphoid tissues by directing lymphocyte egress. In this review, we give an overview of the generation and degradation of S1P in specific lymphoid microenvironments. Furthermore, we discuss, sometimes contradictory, the functions of the five S1P receptors on different cells in diverse tissues and give an idea of additional counteracting chemotactic signals for lymphocyte immigration and emigration. We focus special attention to recent discoveries of S1P-specific transporters, like spinster-homolog-2 and the active secretion of S1P by endothelial cells, erythrocytes and platelets. In addition, we describe the microanatomical structures as well as entry and egress routes into lymphoid organs which lymphocytes use for efficient trafficking. Finally, we give an overview of open questions regarding the regulation of lymphocyte homing from primary lymphoid organs to secondary lymphoid organs and back again.

Immune Regulation by Sphingosine 1-Phosphate and Its Receptors

It is well established that the lysophospholipid and signalling molecule sphingosine 1-phosphate (S1P) has many important functions in immune surveillance. S1P is produced from sphingosine by two distinct sphingosine kinases, SphK1 and SphK2, and acts as an intracellular messenger and as an extracellular ligand of five G protein-coupled cell surface receptors designated S1P(1)-S1P(5). S1P not only regulates peripheral lymphocyte circulation, but also influences their differentiation, activation, infiltration, and local positioning. The therapeutic value of modulating S1P metabolism and S1P receptor function is currently tested in clinical trials and holds great promise for treatment of different autoimmune diseases. Despite its obvious contribution to immune regulation, the analysis of S1P is still challenging. A major obstacle is the difficulty to analyze S1P locally in tissues and within cells due to its high metabolic turnover and the limited resolution of current analytical techniques like liquid chromatography and mass spectrometry. This review focuses on recent advancements to our understanding how different sources of S1P contribute to immune function, and how changes in production, secretion, and degradation of S1P can influence immune responses.

Regulation of metabolism and transport of sphingosine-1-phosphate in mammalian cells

Sphingosine-1-phosphate (S1P), which is generated from the sphingosine kinase-catalyzed phosphorylation of sphingosine, is now recognized as a critical regulator of many kinds of physiological and pathological processes, including cancer, cardiovascular function, and diabetes. It can also trigger a wide variety of biological effect, such as cell movement, differentiation, survival, inflammation, immunity, calcium homeostasis, and angiogenesis. As we know, a number of the biological effects of S1P are mediated by its binding to five specific G protein-coupled receptors located on the cell surface or intracellular targets. However, the synthesis and the secretion of S1P are regulated by various endogenetic or ectogenous stimuli and involve many kinds of enzymes and transporters. In this review, we discuss the regulation of S1P synthesis by many kinds of enzymes and mainly introduce the process of ceramide to S1P. Moreover, S1P deterioration is important balance in physiologic adjustment. We also describe the role of verified or potential transporters in S1P release in detail.

Novel View on Unwanted Side Effects of Radio-Chemotherapy on Bone Marrow (BM) Microenvironment - Radio-Chemotherapy Upregulates BM-Level of Bioactive Lipids, Sphingosine-1- Phosphate (S1P) and Ceramide-1-Phosphate (C1P), That Chemoattract Metastasizing Cancer Cells

Abstract 726 Background:Rhabdomyosracoma (RMS), the most common soft-tissue sarcoma of adolescents and children, frequently infiltrates the BM to such a degree that it often mimics acute lymphoblastic leukemia. The prognosis is poor, particularly for the more aggressive and metastatic alveolar RMS (ARMS) compared to embryonal RMS (ERMS). In our previous work, we demonstrated a pivotal role for two signaling axes, a-chemokine stromal-derived factor-1 (SDF-1)–CXCR4 (a seven-transmembrane-spanning G protein-coupled receptor) and hepatocyte growth factor/scatter factor (HGF/SF)–c-met, in metastasis of pediatric sarcomas to bone marrow (BM) (Blood 2002;100:2597-2606,Cancer Research 2003; 63:7926–7935, IJC 2010;127: 2554–2568). Recently, however, we observed that the bioactive lipids sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) are much more potent chemotractants for human rhabdomyosarcoma (RMS) than SDF-1 or HGF/SF. Importantly, we observed that S1P and C1P levels are highly increased in BM after radio-chemotherapy. Hypothesis: Based on these observations, we hypothesized that S1P and C1P direct chemotaxis of RMS cells to BM. This could be particularly important in patients treated with radio-chemotherapy, where upregulation of S1P and C1P levels in BM may facilitate the spread to the bones of tumor cells that survived initial treatment. Material and Methods: Several complementary in vitro and in vivo approaches were employed to demonstrate a novel role of bioactive lipids in BM metastasis of RMS cells. The expression of S1P seven-transmembrane-spanning G protein-coupled receptors, chemotaxis, adhesion, proliferation, and cell signaling studies in response to S1P and C1P were performed on 8 human ARMS and 3 human ERMS cell lines. The secretion of S1P and C1P in BM and by RMS cells was measured by mass spectrometry (MS). The S1P1 receptor was downregulated by employing an shRNA strategy and S1P1-KO cells were evaluated for their ability to grow tumors in immunodeficient mice. Finally, to address the role of the S1 P–S 1P1 axis in the unwanted spread of sarcoma cells after radio-chemotherapy, we compared seeding of S1P1-KO and control RMS cells in irradiated immunodeficient mice. Results: S1P and C1P are much more potent chemoattractants than SDF-1 or HGF/SF, particularly if employed at “physiological” tissue concentrations. S1P1–5 receptors are expressed on RMS cells and stimulation by S1P induced chemotaxis, adhesion of these cells, and phosphorylation of MAPPp42/44 and AKT. However, while receptor/s for C1P have not yet been identified, C1P also exerted similar effects on human RMS cells. Finally, S1P1-KO cells grew smaller tumors in immunodefcient mice and had impaired seeding efficiency in irradiated animals compared to control RMS cells transduced with empty vector. In parallel experiments, we also observed that both bioactive lipids increase stromalization of the RMS by i) chemoattracting and activating cancer-associated fibroblasts (CAF) and ii) promoting tumor angiogenesis. Conclusions: Both systemic and local radio-chemotherapy leads to upregulation of bioactive lipids in damaged tissues and side effect of such treatment is induction of unwanted prometastatic microenvironment in different organs. By employing an RMS model, we confirmed S1P and identified C1P as novel under-appreciated factor directing metastasis of cancer cells. Since S1P and C1P become upregulated in BM after radio-chemotherapy, both bioactive lipids are involved in the unwanted spread to the bones of RMS cells that survived initial treatment. The role of S1P and C1P in metastasis of other pediatric sarcomas and other types of solid tumors and dissemination of leukemias/lymphomas is currently being investigated in our laboratory, similarly as different strategies to inhibit pro-metastatic effects of S1P and C1P. Disclosures:No relevant conflicts of interest to declare.

Sphingosine kinase 1/S1P pathway involvement in the GDNF‐induced GAP43 transcription

Glial cell line-derived neurotrophic factor (GDNF) is important for the development and maintenance of dopamine neurons (Lin et al. [1993] Science 260: 1130-1132). GDNF is neuroprotective in animal models of Parkinson disease, where dopamine neurons show selective degeneration. We previously reported GDNF-induced SPHK1 gene expression in a neuroblastoma cell line, TGW (Murakami et al. [2007] J Neurochem 102: 1585-1594). In the present study, we focused on the regulatory mechanism of GAP43 (GDNF-induced neuronal phenotype) transcription to further elucidate physiological roles of GDNF-induced SPHK1 expression and activity. Stable wild-type (SPHK1-WT) but not dominant-negative SPHK1 (SPHK1-DN) overexpression increased both control- and GDNF-induced GAP43 expression. SPHK1-WT cells showed enhanced GDNF-induced sphingosine 1-phosphate (S1P) secretion compared with mock- and SPHK1-DN cells. Exogenous S1P also increased GAP43 expression. In TGW cells, PD98059, a MEK inhibitor, but not SB203580 (a p38 MAPK inhibitor) and LY294002 (a PI3K inhibitor) inhibited GDNF-induced GAP43 expression, suggesting the MEK/ERK pathway has a major role in GDNF-induced GAP43 transcription. A G-protein-coupled receptor inhibitor, pertussis toxin, and S1P(1) and S1P(3) receptor antagonists (VPC23019 and CAY10444) also inhibited ERK activation. Moreover, both S1P1 and S1P3 were serine-phosphorylated by GDNF, suggesting their activated states. C/EBPβ transcription factor was induced by GDNF, and DNA pull-down and chromatin immunoprecipitation assays revealed the C/EBP binding site between -131 bp and -98 bp from the first exon of GAP43. Taken together, our results showed that in TGW cells, GDNF increased SPHK1 transcription, leading to the production and secretion of S1P. Through MEK/ERK pathway, S1P stimulates GAP43 transcription with increased binding of C/EBPβ to the 5'-promoter.

Pericyte-Derived Sphinogosine 1-Phosphate Induces the Expression of Adhesion Proteins and Modulates the Retinal Endothelial Cell Barrier

The mechanisms that regulate the physical interaction of pericytes and endothelial cells and the effects of these interactions on interendothelial cell junctions are not well understood. We determined the extent to which vascular pericytes could regulate pericyte-endothelial adhesion and the consequences that this disruption might have on the function of the endothelial barrier. Human retinal microvascular endothelial cells were cocultured with pericytes, and the effect on the monolayer resistance of endothelial cells and expression of the cell junction molecules N-cadherin and VE-cadherin were measured. The molecules responsible for the effect of pericytes or pericyte-conditioned media on the endothelial resistance and cell junction molecules were further analyzed. Our results indicate that pericytes increase the barrier properties of endothelial cell monolayers. This barrier function is maintained through the secretion of pericyte-derived sphingosine 1-phosphate. Sphingosine 1-phosphate aids in maintenance of microvascular stability by upregulating the expression of N-cadherin and VE-cadherin, and downregulating the expression of angiopoietin 2. Under normal circumstances, the retinal vascular pericytes maintain pericyte-endothelial contacts and vascular barrier function through the secretion of sphingosine 1-phosphate. Alteration of pericyte-derived sphingosine 1-phosphate production may be an important mechanism in the development of diseases characterized by vascular dysfunction and increased permeability.

Release of sphingosine‐1‐phosphate from human platelets is dependent on thromboxane formation

Platelets release the immune-modulating lipid sphingosine-1-phosphate (S1P). However, the mechanisms of platelet S1P secretion are not fully understood. The present study investigates the function of thromboxane (TX) for platelet S1P secretion during platelet activation and the consequences for monocyte chemotaxis. S1P was detected using thin-layer chromatography in [(3)H]sphingosine-labeled platelets and by mass spectrometry. Monocyte migration was measured in modified Boyden chamber chemotaxis assays. Release of S1P from platelets was stimulated with protease-activated receptor-1-activating peptide (PAR-1-AP, 100 μM). Acetylsalicylic acid (ASA) and two structurally unrelated reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (500-mg single dose or 100 mg over 3 days) attenuated S1P release from platelets in healthy human volunteers ex vivo. This was paralleled by inhibition of TX formation. S1P release was increased by the TX receptor (TP) agonist U-46619, and inhibited by the TP antagonist ramatroban and by inhibitors of ABC-transport. Furthermore, thrombin-induced release of S1P was attenuated in platelets from TP-deficient mice. Supernatants from PAR-1-AP-stimulated human platelets increased the chemotactic capacity of human peripheral monocytes in a S1P-dependent manner via S1P receptors-1 and -3. These effects were inhibited by ASA-pretreatment of platelets. TX synthesis and TP activation mediate S1P release after thrombin receptor activation. Inhibition of this pathway may contribute to the anti-inflammatory actions of ASA, for example by affecting activity of monocytes at sites of vascular injury.

The analysis of sphingosine 1-phosphate secretion from cells expressing Spns2

The analysis of sphingosine 1-phosphate secretion from cells expressing Spns2

Role of sphingosine kinase localization in sphingolipid signaling

The sphingosine kinases, SK1 and SK2, produce the potent signaling lipid sphingosine-1-phosphate (S1P). These enzymes have garnered increasing interest for their roles in tumorigenesis, inflammation, vascular diseases, and immunity, as well as other functions. The sphingosine kinases are considered signaling enzymes by producing S1P, and their activity is acutely regulated by a variety of agonists. However, these enzymes are also key players in the control of sphingolipid metabolism. A variety of sphingolipids, such as sphingosine and the ceramides, are potent signaling molecules in their own right. The role of sphingosine kinases in regulating sphingolipid metabolism is potentially a critical aspect of their signaling function. A central aspect of signaling lipids is that their hydrophobic nature constrains them to membranes. Most enzymes of sphingolipid metabolism, including the enzymes that degrade S1P, are membrane enzymes. Therefore the localization of the sphingosine kinases and S1P is likely to be important in S1P signaling. Sphingosine kinase localization affects sphingolipid signaling in several ways. Translocation of SK1 to the plasma membrane promotes extracellular secretion of S1P. SK1 and SK2 localization to specific sites appears to direct S1P to intracellular protein effectors. SK localization also determines the access of these enzymes to their substrates. This may be an important mechanism for the regulation of ceramide biosynthesis by diverting dihydrosphingosine, a precursor in the ceramide biosynthetic pathway, from the de novo production of ceramide.